Methadone, phencyclidine, and their metabolites were extracted from plasma and separated on a high-performance liquid chromatographic (HPLC) column using the fluorescent 9,10-dimethoxyan- thracene-2-sulfonic acid as a counterion. The chromatographed mobile phase was subsequently extracted on-line with chloroform. The separated organic phase, containing the fluorescent ion-pairs of the investigated amines, was analyzed in the flow cell of a fluorometer (excitation 380nm, emission 445nm). The phase separator volume was as small as possible to avoid dead volume. The method was also applied to the bioassay of cocaine with a sensitivity of 1–6ng/ml of plasma. Application of these assays gave a red blood cell-plasma water partition coefficient for-methadone of 3.39±0.26 (SD) in a concentration range up to 20 μg/ml, and demonstrated a time-dependent partition with a diffusion half-life of 1.44min±0.26min (SD). The protein binding of methadone determined by ultracentrifugation was concentration dependent and varied between 75-62% at the highest concentration studied (9 μg/ml). The presence of the major metabolite did not have any influence on the protein binding. The results were confirmed by using the red blood cell- partitioning method to determine the protein binding.